CN108060158A - A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead - Google Patents
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead Download PDFInfo
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- CN108060158A CN108060158A CN201810138156.4A CN201810138156A CN108060158A CN 108060158 A CN108060158 A CN 108060158A CN 201810138156 A CN201810138156 A CN 201810138156A CN 108060158 A CN108060158 A CN 108060158A
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Abstract
The invention discloses the preparation methods that a kind of chitosan magnetic carries denitrifying bacteria bead, belong to environment remediation field, which is characterized in that comprise the following steps：A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The chitosan mixed solution of ion；B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is freeze-dried after crosslinking, forms chitosan magnetic bead；C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, it obtains chitosan magnetic and carries denitrifying bacteria bead.The present invention can enter water body as the growth and breeding of denitrifying bacteria slowly releases denitrifying bacteria thalline, and the nitrate pollutants in water are effectively removed, and slow release effect is preferable, not generate secondary pollution, and product has magnetism, easily recycles and manufacturing cost is low.
Description
Technical field
The present invention relates to groundwater environmental repairing fields more particularly to a kind of chitosan magnetic to carry denitrifying bacteria bead
Preparation method.
Background technology
Groundwater azotate pollution problem and human health are closely related, often drink the water meeting of the nitrate containing higher concentration
Human body is made to suffer from methemoglobinemia due to anoxic.At present, for azotate pollution in water, main minimizing technology has abiotic
Method and bioanalysis.Nonbiological method generally existing is of high cost, removal is not thorough, has the shortcomings that secondary pollution.Bioanalysis is to utilize certainly
Denitrification makes nitrate nitrogen be eventually converted into N under microbial action existing for right boundary2O or N2Process, with nonbiological method
It compares, there is removal is thorough, processing cost is low etc..
But free denitrifying bacteria, when handling azotate pollution underground water, there are following four shortcomings.When with
Being continuously increased for bacterial number, its cluster is caused easily to sink to water bottom so that nitrate removal rate reduction；It is second is that free
There is the shortcomings that not easy to maintain in microorganism；Third, free microorganism can cause its loss because of factors such as environment, Competitions；Four
Be free bacterium in actual application, be not easily recycled recycling.
Chitosan magnetic bead is a kind of new functional macromolecule material developed in recent years, is wrapped up with chitosan
The particle with chondritic that magnetic particle is formed.Chitosan magnetic bead have prepare it is simple, of low cost and without life
The characteristics of object toxicity, the amino on surface can be with the carboxyl reactions of protein molecule, so as to by protein adsorption；Surface
Hydroxyl can be combined with bacteria cell wall, therefore, bacterium is able to load stabilization.Meanwhile it is adsorbed using chitosan magnetic bead
It is proved in water in the circulation experiment of heavy metal, chitosan magnetic bead has good physical and chemical stability in itself.To up to the present
It is a kind of permeable reaction wall filler with potential advantages for in-situ treatment groundwater pollutant afterwards.
Publication No. CN 103803704A, publication date disclose one kind for the Chinese patent literature of on May 21st, 2014
For the preparation method of the permeable reaction dielectric material of groundwater azotate pollution[9], including：(1) in mass ratio it is 1-3:
0.1-0.5:1-3：1.5-3:0.5-1:0.5-1.5:50 ratios take quartz sand, denitrifying bacteria, chitosan, CaCl2, alginic acid
Sodium, cladding nanometer iron and pH are 7 Na2PO4-NaH2PO4Buffer solution；(2) by sodium alginate, chitosan, quartz sand and
Na2HP04-NaH2PO4Buffer solution mixing under 30-40 DEG C of constant temperature, obtains mixed liquor, is cooled to room temperature；(3) cladded type is received
Meter Tie and denitrifying bacteria are added in mixed liquor, are uniformly mixed, and are made and are granulated slurries；(4) CaCl that will have been taken2Solid is molten
In pure water, the CaCl that mass concentration is 3-6% is made2Cross-linking agent aqueous solution；(5) the granulation slurries of step 3 are added drop-wise to step 4
CaCl2It in cross-linking agent aqueous solution, is crosslinked at room temperature, spheric granules is made；(6) spheric granules is cleaned, at room temperature wind
It is dry, obtain target product.
The preparation method of the disclosed permeable reaction dielectric material for groundwater azotate pollution of the patent document,
PH value applicability is poor in application process, and redox reaction, which occurs, for nanometer Fe O and the nitrate of cladding can generate secondary dirt
Contaminate object；The growth and breeding situation of microorganism does not reflect that denitrification experience duration is also indefinite more preferably.
The content of the invention
The defects of present invention is in order to overcome the above-mentioned prior art provides a kind of chitosan magnetic and carries denitrifying bacteria bead
Preparation method, in of the invention, microorganism and chitosan magnetic bead surface hydroxy combining, fixing-stable are carried out using pollutant
The growth and breeding of itself constantly carries out microorganism replacement, slowly releases denitrifying bacteria thalline and enter polluted-water, in water
Nitrate pollutants effectively removed, slow release effect is preferable, does not generate secondary pollution, and product has easily separated time magnetic
It receives, it is reusable, manufacturing cost is low, and with the application prospect as permeable reaction wall filler.
The present invention is achieved through the following technical solutions：
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, which is characterized in that comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.1-0.3, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 25-35 DEG C to carry temperature, and the pH of culture solution is 7-9, and nitrate nitrogen concentration is 25-75mg/l in culture solution, continuously cultivates duration
For 120h.
Beneficial effects of the present invention are mainly manifested in following aspect：
First, it is of the invention, " Fe a, is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+
The chitosan mixed solution of ion；B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise,
It is freeze-dried after crosslinking, forms chitosan magnetic bead；C, chitosan magnetic bead is put into added with denitrifying bacteria bacterium
It is continuous to cultivate in the culture solution of liquid, it obtains chitosan magnetic and carries denitrifying bacteria bead." using obtained by a-c particular steps
Chitosan magnetic carries denitrifying bacteria bead, as the biological material for removing nitrate in water removal, can effectively remove nitre in water
Hydrochlorate, pH accommodations are wider, utilize the growth and breeding of microorganism itself so that thalline is sustained, while material has in itself
It is magnetic, its aggregation can be made by externally-applied magnetic field, is easily recycled, and product is N2And H2O, environmentally friendly, non-secondary pollution
Object generates, and product has a magnetic easily separated recycling, reusable, manufacturing cost is low, and with as permeable reaction wall
The application prospect of filler.
2nd, it is of the invention, in step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2%
Stirring and dissolving obtains in the sour environment of glacial acetic acid, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through
FeCl3·6H2O dissolves acquisition in water, and amino content is then bigger in the higher chitosan molecule of degree of deacetylation, active site
It is more, be more conducive to Fe2+And Fe3+The coordination of ion.
3rd, it is of the invention, in step a, chitosan solution and Fe2+And Fe3+The speed of agitator of solion mixing is specifically controlled
It makes in 300r/min, is because rotating speed is excessive, mixes the chitosan and Fe of completion2+And Fe3+It is present with a large amount of gas in solion
Bubble, easily makes b step generate the chitosan magnetic bead of hangover, and rotating speed controls in 300r/min, then can effectively avoid trailing
Chitosan magnetic bead generate；Incorporation time is 30min, and the time, too short chitosan was to Fe3+Ion and Fe2+Cannot fully it match somebody with somebody
Position, overlong time, the Fe in solution2+It can be fully oxidized because of the oxygen in air as Fe3+。
4th, it is of the invention, in step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline immersion dropwise
Liquid refers to that by peristaltic pump Fe will be contained under the rotating speed of 1r/min2+And Fe3+The chitosan mixed solution of ion instills alkalescence leaching
It steeps in liquid, is crosslinked the crosslinking agent used as sodium citrate, a length of 10h during crosslinking, if crosslinking time is too short, nanometer Fe3O4Particle is given birth to
Into incomplete, the physics and chemical stability of bead are influenced.
5th, of the invention, in step b, material is freezed, its moisture content contained is made to become ice by a length of 36h during freeze-drying,
Then under vacuo ice is made to distil, dried material keeps original chemical composition and physical property, and from surface tension
Effect, sample is indeformable.
6th, it is of the invention, in step c, the chitosan magnetic bead prepared is put into the nutrient solution added with bacterium solution and is carried out
Load, maintains the original activity of microorganism, does not influence the metabolic process of its own, and the growth and breeding mistake for passing through its own
Journey achievees the purpose that load, and loading process is simple, easy to operate.
Description of the drawings
The present invention is described in further detail below in conjunction with specification drawings and specific embodiments：
Fig. 1 is the scanning electron microscope image of the chitosan magnetic bead of unsupported denitrifying bacteria；
Fig. 2 is the scanning electron microscope image for the chitosan magnetic bead for being loaded with denitrifying bacteria；
Fig. 3 is the variation that chitosan magnetic carries three nitrogen in denitrifying bacteria bead denitrification process in the embodiment of the present invention 6
Schematic diagram；
Fig. 4 is that chitosan magnetic carries 0D in denitrifying bacteria bead denitrification process in the embodiment of the present invention 6600Variation
Schematic diagram.
Specific embodiment
It, below will be to the technical side in the embodiment of the present invention to make the object of the invention, technical solution and advantage clearer
Case is clearly and completely described.The person that is not specified actual conditions in embodiment, carries out according to normal condition.Agents useful for same or instrument
Production firm person is not specified in device, is the conventional products that can be obtained by commercially available purchase.
Main agents：Chitosan, wherein deacetylation >=90%, viscosity 100-200mpas, glacial acetic acid, hydroxide
Sodium, sodium citrate, sodium nitrate, FeCl3·6H2O and FeCl2·4H2O, selected from section's dragon chemical reagent factory.All compounds are
Commercially available chemicals, without being further purified.Ultra-pure water used in all experiments is Milli-Q water purification systems Ulupure
Company generates.
Key instrument:PHS-320 acidometers are using Chengdu ShiJiFangZhou Science and Technology Ltd., JBXL-70 water-baths vibration
Case is the manufacture of Changzhou Pu Tian instrument manufacturings Co., Ltd, BSA224S-CW electronic balances are Sai Duolisi scientific instrument Co., Ltd
Manufacture, F series freeze driers are manufactured by NingBo XinZhi Biology Science Co., Ltd, V-1100D visible spectrophotometers
It is manufactured by the U.S. spectrum in Shanghai up to Instrument Ltd..
Embodiment 1
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.1, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 25 DEG C to carry temperature, and the pH of culture solution is 7, and nitrate nitrogen concentration is 25mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 25mg/l, pH are 7, and at 25 DEG C, nitrate removal rate is 100%, and a length of 144 is small during experience
When, at the end of bacterium solution OD600For 0.084, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 2
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.2, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 25 DEG C to carry temperature, and the pH of culture solution is 7, and nitrate nitrogen concentration is 25mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 25mg/l, pH are 7, and at 25 DEG C, nitrate removal rate is 100%, and a length of 143 is small during experience
When, at the end of bacterium solution OD600For 0.087, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 3
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.3, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 25 DEG C to carry temperature, and the pH of culture solution is 7, and nitrate nitrogen concentration is 25mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 25mg/l, pH are 7, and at 25 DEG C, nitrate removal rate is 100%, and a length of 140 is small during experience
When, at the end of bacterium solution OD600For 0.091, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 4
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.1, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 30 DEG C to carry temperature, and the pH of culture solution is 8, and nitrate nitrogen concentration is 50mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 50mg/l, pH are 8, and at 30 DEG C, nitrate removal rate is 100%, and a length of 122 is small during experience
When, at the end of bacterium solution OD600For 0.094, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 5
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.2, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 25 DEG C to carry temperature, and the pH of culture solution is 8, and nitrate nitrogen concentration is 50mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 50mg/l, pH are 8, and at 30 DEG C, nitrate removal rate is 100%, and a length of 120 is small during experience
When, at the end of bacterium solution OD600For 0.097, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 6
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.3, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 30 DEG C to carry temperature, and the pH of culture solution is 8, and nitrate nitrogen concentration is 50mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 50mg/l, pH are 8, and at 30 DEG C, nitrate removal rate is 100%, and a length of 120 is small during experience
When, at the end of bacterium solution OD600For 0.103, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 7
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.1, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 35 DEG C to carry temperature, and the pH of culture solution is 9, and nitrate nitrogen concentration is 75mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 75mg/l, pH are 9, and at 35 DEG C, nitrate removal rate is 100%, and a length of 135 is small during experience
When, at the end of bacterium solution OD600For 0.063, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 8
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.2, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 35 DEG C to carry temperature, and the pH of culture solution is 9, and nitrate nitrogen concentration is 75mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 75mg/l, pH are 9, and at 35 DEG C, nitrate removal rate is 100%, and a length of 134 is small during experience
When, at the end of bacterium solution OD600For 0.068, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Embodiment 9
A kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The shell of ion gathers
Sugared mixed solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, is carried out after crosslinking cold
It is lyophilized dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, obtain magnetism
Chitosan-loaded denitrifying bacteria bead.
In the step a, chitosan solution by the chitosan of deacetylation >=90% in volume fraction be 2% glacial acetic acid
Sour environment in stirring and dissolving obtain, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·
6H2O dissolves acquisition in water.
It is 600r/min that the chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and when stirring is a length of
30min, temperature are room temperature.
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed is
300r/min, temperature are room temperature.
In the step b, the alkalescence soak is configured by NaOH and sodium citrate.
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to dropwise
Fe will be contained under the rotating speed of 1r/min by peristaltic pump2+And Fe3+The chitosan mixed solution of ion instills alkaline soak
In.
In the step b, the crosslinking agent used is crosslinked as sodium citrate, and a length of 10h during crosslinking, when freeze-drying is a length of
36h。
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is born
It carries, OD600For 0.3, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, is born
It is 35 DEG C to carry temperature, and the pH of culture solution is 9, and nitrate nitrogen concentration is 75mg/l in culture solution, continuously a length of 120h during culture.
Chitosan magnetic carries the application of denitrifying bacteria bead, and chitosan magnetic load denitrifying bacteria bead is put into nitric acid
Salt nitrogen concentration is in the nutrient solution that 75mg/l, pH are 9, and at 35 DEG C, nitrate removal rate is 100%, and a length of 130 is small during experience
When, at the end of bacterium solution OD600For 0.072, at the end of accumulated without ammonia nitrogen and nitrite nitrogen.
Below by specific experiment by chitosan magnetic bead, sodium alginate embedding denitrifying bacteria after gelled pill with
Chitosan magnetic prepared by the present invention carries denitrifying bacteria bead and carries out comparative illustration：
Chitosan magnetic is carried denitrifying bacteria bead, chitosan magnetic bead, sodium alginate gel bead respectively to be put into
Nitrate nitrogen concentration is 50mg/l, OD600For 0.1-0.3, pH is in 8 nutrient solution, and at 30 DEG C, concussion carries out nitrate
Removal.Acquired results are as shown in table 1 below：
Table 1
It is uneven to can be seen that the chitosan magnetic bead surface of unsupported microorganism from Fig. 1 and Fig. 2, after load,
It is basic at bead surface bumps to disappear, illustrate that by preparation method of the present invention bacterium success on chitosan magnetic bead can be made
Load.
With reference to Fig. 3 and Fig. 4, as can be drawn from Table 1, under the same conditions, simple chitosan magnetic bead is to nitre in water
Hydrochlorate acts on substantially without removal.In addition, chitosan magnetic carries denitrifying bacteria bead and is loaded with the sodium alginate of denitrifying bacteria
Gelled pill reaches 100% to the removal rate of nitrate, and ammonia nitrogen and nitrite nitrogen are not detected after denitrification.
Unlike, during preparing chitosan magnetic and carrying denitrifying bacteria bead, maintain the activity of microorganism so that removal
During nitrate, the reaction time is shorter, and microbial growth is more rapid.Thus show that chitosan magnetic carries anti-nitre
Change Bacterial pellets in preparation process to the activity influence smaller of microorganism, it is rapider to the removal of nitrate nitrogen, more effectively.
Claims (10)
1. a kind of chitosan magnetic carries the preparation method of denitrifying bacteria bead, which is characterized in that comprises the following steps：
A, Fe is added into chitosan solution2+And Fe3+Ion stirs and evenly mixs to be formed containing Fe2+And Fe3+The chitosan of ion mixes
Close solution；
B, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak dropwise, and it is dry that freezing is carried out after crosslinking
It is dry, form chitosan magnetic bead；
C, chitosan magnetic bead is put into the culture solution added with denitrifying bacteria bacterium solution, it is continuous to cultivate, it obtains magnetic crust and gathers
Sugar carries denitrifying bacteria bead.
2. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step a, chitosan solution is by the chitosan of deacetylation >=90% in the acyclic acidic that volume fraction is 2% glacial acetic acid
Stirring and dissolving obtains in border, Fe2+Pass through FeCl2·4H2O dissolves acquisition, Fe in water3+Ion passes through FeCl3·6H2O is in water
Dissolving obtains.
3. a kind of chitosan magnetic according to claim 2 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
It is 600r/min that chitosan, which is dissolved in the mixing speed that volume fraction is 2% glacial acetic acid, and a length of 30min during stirring, temperature is room
Temperature.
4. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step a, Fe is added2+And Fe3+Ion, Fe2+And Fe3+The molar ratio of ion is 1:2, mixing speed 300r/min, temperature
It spends for room temperature.
5. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step b, alkaline soak is formulated by NaOH and sodium citrate.
6. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step b, Fe will be contained2+And Fe3+The chitosan mixed solution of ion instills alkaline soak and refers to pass through wriggling dropwise
Pump will contain Fe under the rotating speed of 1r/min2+And Fe3+The chitosan mixed solution of ion is instilled in alkaline soak.
7. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step b, the crosslinking agent used is crosslinked as sodium citrate, a length of 10h during crosslinking, a length of 36h during freeze-drying.
8. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step b, chitosan magnetic bead is repeatedly rinsed with deionized water, cleans remaining alkali and crosslinking agent.
9. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, it is characterised in that：
In the step c, the chitosan magnetic bead prepared is put into the nutrient solution added with 30ml bacterium solutions and is loaded, OD600For
0.1-0.3, OD600It is measured with 10mm cuvettes under 600nm wavelength by visible spectrophotometer.
10. a kind of chitosan magnetic according to claim 1 carries the preparation method of denitrifying bacteria bead, feature exists
In：In the step c, continuous culture refers to be carried out in the dark in water-bath concussion case, and concussion speed is 160r/min, load temperature
For 25-35 DEG C, the pH of culture solution is 7-9, and nitrate nitrogen concentration is 25-75mg/l in culture solution, a length of 120h during continuous culture.
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| CN115490316A (en) * | 2022-01-24 | 2022-12-20 | 成都理工大学 | Reusable underground water nutrient substance slow-release capsule body |
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